This invention relates to immunostimulating complex (ISCOM) matrices prepared using saponin preparations, particularly saponin preparations derived from the bark of Quillaja saponaria (Molina). The invention also extends to immunogenic immunostimulating complexes in which immunogens are incorporated into an immunostimulating complex matrix in accordance with this invention. Such immunogens may be proteins or polypeptides derived from bacteria, viruses or other microorganisms, but they may, in addition, be synthetic, particularly recombinant proteins, or polypeptides which can induce an immune response.
The adjuvant properties of saponin have been long known, as has its abiiity to increase antibody titres to immunogens. As used herein, the term xe2x80x9csaponinxe2x80x9d refers to a group of surface-active glycosides of plant origin composed of a hydrophilic region (usually several sugar chains) in association with a hydrophobic region of either steroid or triterpenoid structure. Although saponin is available from a number of diverse sources, saponins with useful adjuvant activity have been derived from the South American tree Quillaja saponaria (Molina). Saponin from this source was used to isolate a xe2x80x9chomogeneousxe2x80x9d fraction denoted xe2x80x9cQuil Axe2x80x9d (Dalsgaard, 1974).
Dose-site reactivity is a major concern for both the veterinary and human use of Quil A in vaccine preparations. One way to avoid this toxicity of Quil A is the use of immunostimulating complexes (known as ISCOMs, an abbreviation for Immuno Stimulating COMplexes). This is primarily because Quil A is less reactive when incorporated into immunostimulating complexes, because its association with cholesterol in the complex reduces its ability to bind to cholesterol in cell membranes and hence its cell lytic effects. In addition, a lesser amount of Quil A is required to generate a similar level of adjuvant effect. Immunostimulating complexes are small, cage-like structures 30 to 40 nm in diameter which retain this structure on freeze drying. The size can vary however depending on mode of preparation, composition and the method used for measurement. The final formulation of a typical immunostimulating complex with an optimal amount of immunogenic protein or polypeptide is a weight ratio of Quil A, cholesterol, phospholipids, and protein or polypeptide (5:1:1:1). Such a typical immunostimulating complex is estimated to contain around 60% by weight Quil A, around 10% each for cholesterol and phospholipids, and the remainder protein or polypeptide. Proteins or polypeptides can be incorporated into the immunostimulating complex matrix either directly or by chemical coupling to a carrier protein (e.g. influenza envelope protein) after incorporation of the carrier protein into the immunostimulating complex.
As an adjuvant, the immunostimulating complex matrix confers many advantages including powerful immunostimulatory effects, low toxicity, ability to induce both cellular (including CTL) and humoral responses, and it is inexpensive in both reagent and manufacturing cost. However, in the past, immunostimulating complexes have had two major disadvantages; firstly, the Quil A used in their preparation was a complex and ill-defined mixture of a biologically-derived product, and batch-to-batch variation was therefore to be expected; and secondly, the complexes still showed reduced but measurable haemolytic activity which could be expected to indicate a certain level of dose-site reactivity.
Since the recognition of the adjuvant activity of Quil A (Dalsgaard, 1974) several groups have further fractionated this material into a number of xe2x80x9cpurifiedxe2x80x9d components (Australian Patent Specification No. 632067; Kersten, 1990; Kensil, 1988; Kensil 1991). These components were subsequently shown to have variable properties especially in regards to adjuvant activity, haemolytic activity and ability to form immunostimulating complexes. The use of defined or purified saponin components conferred two potential advantages for their use in a human vaccine. Firstly, these components could be characterised and therefore made reproducibly; and secondly, the components could be selected for optimal usefulness.
The immunomodulatory properties of the Quil A saponins and the additional benefits to be derived from these saponins when they are incorporated into an immunostimulating complex have been described in various publications, e.g. Cox and Coulter, 1992; Dalsgaard, 1974; Morein et al., Australian Patent Specifications Nos. 558258, 589915, 590904 and 632067. In Australian Patent Specification No. 632067, the separation of a preparation of Quil A into three distinct fractions called B4b, B3 and B2 is described, along with HPLC chromatographic procedures for this fractionation.
One of the most useful methods for producing recombinant proteins or polypeptides for vaccine (and other) purposes relies on the incorporation of a metal-chelating sequence (usually polyhistidine) at the N- or C-terminus of the recombinant product. This allows facile purification of the product by Immobilized Metal Affinity Chromatography (IMAC), and is especially useful in cases where the protein or polypeptide requires the presence of strong denaturants (such as urea) for solubility (Porath, 1992). Such proteins or polypeptides, however, are difficult to formulate as vaccines using immunostimulating complex (ISCOM) technology. This is because:
1. in many cases removal of the denaturant results in precipitation of the protein or polypeptidexe2x80x94the alternative is not to remove the denaturant, however this may result in unacceptable toxicity of the vaccine or poor stability of vaccine formulations; and
2. it is difficult to efficiently incorporate such proteins or polypeptides in immunostimulating complex matrices, since this requires that the protein or polypeptide be ampipathic in characterxe2x80x94this is a property of proteins that span cell membranes, but very few other proteins.
Incorporation of proteins or polypeptides into immunostimulating complex has two major benefits:
1. the extremely hydrophilic character of the matrix particle will prevent the precipitation of hydrophobic proteins or polypeptides that are insoluble in the absence of denaturant; and
2. incorporation of the protein or polypeptide into immunostimulating complexes will provide the maximum adjuvant effect. This is especially the case for cytotoxic T-lymphocyte responses, where codelivery of saponin and immunogen to the same antigen-presenting cell may be an absolute requirement for obtaining an adequate immune response. Clearly, codelivery will be much more efficient if the protein or polypeptide is anchored in an immunostimulating complex.
It is an object of the present invention to provide a simple and effective method for incorporating a protein or polypeptide, particularly a metal-chelating protein or polypeptide, into immunostimulating complex matrix particles.
Shnek et al. (1994) disclose a method for targeting proteins to lipid assemblies known as liposomes using a phospholipid-like molecule in which the head group is a chelating iminodiacetic acid (IDA), and show that such liposomes can bind histidine-rich myoglobin in the presence of metal ions.
According to the present invention, there is provided an immunostimulating complex matrix comprising a saponin preparation, a sterol and a phospholipid, said matrix further comprising a metal-chelating moiety capable of binding a protein or polypeptide having at least one chelating amino acid sequence in the presence of metal ions.
In another aspect, the present invention also provides an immunogenic immunostimulating complex which comprises a matrix as broadly described above and an immunogenic protein or polypeptide having at least one chelating amino acid sequence, said protein or polypeptide being bound to said matrix in the presence of metal ions.